As genome sequencing becomes increasingly routine, the ability to genetically manipulate organisms of interest has become indispensable in order to rigorously parse gene function. Whereas commonly used model organisms often have robust (although sometimes limited) options for controlling gene expression, these techniques are not always applicable in less commonly studied organisms. Meissner et al., “Molecular tools for analysis of gene function in parasitic microorganisms” Appl Microbiol Biotechnol 75:963-975 (2007). For example, RNA interference is only useful in cells possessing the requisite machinery. Hammond S. M., “Dicing and slicing: the core machinery of the RNA interference pathway” FEBS Lett 579:5822-5829 (2005); and Hannon G. J., “RNA interference” Nature 418:244-251 (2002). Further, transcriptional control requires detailed knowledge of protein components, regulatory sequences, and promoter architecture, which are often evolutionarily divergent. Borneman et al., “Divergence of transcription factor binding sites across related yeast species” Science 317:815-819 (2007); and Odom et al., “Tissue-specific transcriptional regulation has diverged significantly between human and mouse” Nat Genet 39:730-732 (2007). Techniques to make knockouts are sometimes available but are not always adaptable to studying essential genes. Furthermore, the study of newly discovered organisms would benefit significantly from experimenter-controlled regulatory systems that obviate extensive upfront characterization of their particular genetics. Beyond elucidating basic biology, a larger set of genetic control techniques will be required for building complex intracellular circuits, as is vital to the field of synthetic biology.
Since many mechanistic details of protein translation are highly conserved across different genera, protein translation is an attractive platform for designing modular, inducible gene expression systems. Bruell et al., “Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis” Biochemistry 47:8828-8839 (2008); and Kapp et al., “The molecular mechanics of eukaryotic translation” Annu Rev Biochem 73:657-704 (2004). In principle, these systems can be readily implemented in a wide variety of contexts. Nature provides numerous examples of post-transcriptional regulation, including: i) antisense (Good L., “Translation repression by antisense sequences” Cell Mol Life Sci 60:854-861 (2003)); ii) attenuation (Kolter et al., “Attenuation in amino acid biosynthetic operons” Annu Rev Genet 16:113-134 (1982); and iii) RNA interference (Mello et al., “Revealing the world of RNA interference” Nature 431:338-342 (2004)).
What is needed in the art are broadly-applicable methods to specifically control protein translation in a desired organism or cell-free biological system by the presence or absence of endogenous and/or exogenous ligands.